Use of transplatin to prevent hearing loss

ABSTRACT

Methods and compositions for treating and preventing toxic side effects of platinum-based chemotherapy agents are disclosed, in which transplatin is administered to a subject. Transplatin is shown to have protective effects against cisplatin-induced ototoxicity, nephotoxicity and neurotoxicity. Anti-inflammatory activity of transplatin is demonstrated and methods and compositions for treating and preventing inflammatory pain are described.

RELATED APPLICATION INFORMATION

The present application claims priority to U.S. Application No.61/225,772 filed Jul. 15, 2009 and U.S. Non-provisional patentapplication Ser. No. 12/836,398 filed Jul. 14, 2010, the entiredisclosures of which are herein incorporated by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numberDC009950 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

TECHNICAL FIELD

The present disclosure relates generally to platinum-based chemotherapyagents, and in particular to methods and compositions for treating orpreventing side effects of platinum-based chemotherapy agents in cancertreatment, and to treat or prevent inflammatory pain.

BACKGROUND OF THE INVENTION

Cisplatin is a highly effective chemotherapeutic agent and widely usedfor treating solid tumors. However, cisplatin therapy produces seriousside effects such as nephrotoxicity, neurotoxicity, and severeototoxicity. In particular, cisplatin-induced ototoxicity is bilateraland irreversible, and thus particularly serious in the pediatricpopulation and especially during early development to about three yearsof age. Cisplatin is frequently used to treat cancers like neuroblastomaand CNS neuroblastoma tumors in pediatric patients, but the resultingototoxicity hampers speech, cognition and social development. Thus, agreat need exists for treatments that will ameliorate cisplatin-inducedototoxicity and other side effects in children but also in adults.Carboplatin and oxaliplatin are two other platinum-based chemotherapyagents that have emerged over the last twenty years of intense researchaimed at improving cisplatin. Unfortunately, carboplatin and oxaliplatinare effective in treating only a few cancers. Moreover, whileoxaliplatin has been used to treat children with central nervous system(CNS) tumors, it has very limited activity (M. Fouladi et al., Phase IIstudy of oxaliplatin in children with recurrent or refractorymedulloblastoma, supratentorial primitive neuroectodermal tumors, andatypical teratoid rhabdoid tumors: a pediatric brain tumor consortiumstudy, CANCER 107:2291-2297 (2006)), and also consistently producesneuropathies (L. M. Pasetto et al., Oxaliplatin-related neurotoxicity:how and why? CRIT. REV. ONCOL. HEMATOL. 59:159-68 (2006)).

Transplatin (trans-diamminedichloroplatinum (II)), the transstereoisomer of cisplatin, has the formula trans-[PtCl₂(NH₃)₂] andthough well-studied does not exhibit a comparably useful pharmacologicaleffect, though it has been shown to be relatively nontoxic in humans.Although ineffective as an antitumor agent (Coluccia, M., and Natile,G., Trans-platinum complexes in cancer therapy, ANTICANCER AGENTS MED.CHEM. 7:111-123 (2007)), transplatin does not cause ototoxicity atequimolar doses of cisplatin. Its low activity is generally thought tobe due to rapid deactivation of the agent before it can interact withthe DNA. It is known for instance that transplatin stereochemistry doesnot allow it to form intrastrand DNA cross-links (Pinto, A. L., andLippard, S. J., Sequence-dependent termination of in vitro DNA synthesisby cis- and trans-diamminedichloroplatinum (II), PROC. NATL. ACAD. SCI.USA 82:4616-4619 (1985)) formed by cisplatin (cis-DDP). Intrastrandcrosslinking of DNA by cisplatin is thought to be the primary DNA adductunderlying cytotoxicity (A. C. Plooy et al., Induction and repair of DNAcross-links in chinese hamster ovary cells treated with various platinumcoordination compounds in relation to platinum binding to DNA,cytotoxicity, mutagenicity, and antitumor activity, CANCER RES.44:2043-2051 (1984)). Cis- and transplatin demonstrate differentialDNA-adduct formation, DNA-protein binding and repair mechanisms (R. B.Ciccarelli et al, In vivo effects of cis- andtrans-diamminedichloroplatinum (II) on SV40 chromosomes: differentialrepair, DNA-protein cross-linking, and inhibition of replication,BIOCHEMISTRY 24:7533-7540 (1985)).

Importantly, transplatin rapidly binds to DNA, reaches a maximum at 6hours, then rapidly decreases over the next 6 hours and continues todecrease over the next 36 hours. In contrast, cisplatin bound DNAcontinues to increase steadily over 48 hours. For example, it has beenshown that the level of cisplatin bound to cellular DNA is approximately11-fold higher than that of transplatin over a treatment period of 42hours using equal extracellular concentrations (10 μM) of both theisomers. (T. Saito et al, Similar pharmacokinetics and differentialototoxicity after administration with cisplatin and transplatin inguinea pigs, ACTA LARYNGOL. 117:61-65 (1997)).

A need remains for new approaches to treating or preventing the toxicside effects of effective anti-cancer agents, including thoseplatinum-based chemotherapy agents which are highly effective in cancertherapy, such as cisplatin.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides a pharmaceuticalcomposition for treating cancer in a subject in need thereof, thepharmaceutical composition comprising transplatin and a secondplatinum-based chemotherapy agent. The second platinum-basedchemotherapy agent can be, for example, cisplatin, carboplatin,oxaliplatin, nedaplatin or nanoplatin. The pharmaceutical compositionmay further include a pharmaceutically acceptable carrier, excipient ordiluent. The pharmaceutical composition may further include anon-steroidal anti-inflammatory agent. In the composition, the amount ofthe second platinum-based chemotherapy agent can be between about 0.1and about 100 times the weight of transplatin. The amount of transplatincan be between about 0.1 mg/kg and about 20 mg/kg, preferably betweenabout 0.1 and about 5 mg/kg.

In another aspect, the present disclosure provides a method of treatingor preventing ototoxic effects of an ototoxic agent in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of transplatin. The method can includefor example administering the transplatin to the subject before orduring exposure of the subject to the ototoxic agent. Alternatively, inthe method the transplatin may be administered to the subject afterexposure of the subject to the ototoxic agent. The transplatin can beadministered for example via intraperitoneal injection or trans-tympanicinjection.

In another aspect, the present disclosure provides a method of treatingor preventing nephrotoxic effects of a nephrotoxic agent in a subject inneed thereof, the method comprising administering to the subject atherapeutically effective amount of transplatin. The method can includefor example administering the transplatin to the subject before orduring exposure of the subject to the nephrotoxic agent. Alternatively,in the method the transplatin may be administered to the subject afterexposure of the subject to the nephrotoxic agent. The transplatin can beadministered for example via intraperitoneal injection.

In another aspect, the present disclosure provides a pharmaceuticalcomposition for treating or preventing inflammatory pain in a subject inneed thereof, wherein the pharmaceutical composition comprises atherapeutically effective amount of transplatin. A method of treating orpreventing inflammatory pain in a subject in need thereof is alsoprovided and can include administering to the subject such apharmaceutical composition. In such a method, the transplatin can beadministered via intraperitoneal injection, subcutaneous injection, orintraganglionic injection. The method can be used to treat a subjectsuffering from an inflammatory pain condition, such as for examplearthritis or cancer-induced pain. The method can be used to treat asubject suffering chronic inflammatory pain.

In another aspect, the present disclosure provides a method of treatingor preventing platinum-based chemotherapy agent-induced toxicity in asubject to be treated or treated with a platinum-based chemotherapyagent that is not transplatin, in which a therapeutically effectiveamount of transplatin is administered to the subject. In the method, thetransplatin may be administered to the subject before or during exposureof the subject to the platinum-based chemotherapy agent. Alternatively,the transplatin may be administered to the subject after exposure of thesubject to the platinum-based chemotherapy agent. The platinum-basedchemotherapy agent and transplatin may be administered to the subjecttogether in a pharmaceutical composition, which may further include apharmaceutically acceptable carrier, excipient or diluent.Alternatively, the platinum-based chemotherapy agent and transplatin canbe administered to the subject in separate pharmaceutical compositions.These may be administered simultaneously to the subject, or sequentiallyto the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing in color.Copies of this patent or patent application publication with colordrawings will be provided by the Office upon request and payment of thenecessary fee.

FIG. 1 is a schematic diagram of the interaction between TRPV1 and NOX3in the induction of hair cell damage or death.

FIG. 2A is a panel of photomicrographs of UB/OC-1 cells treated withcisplatin (2.5 μM) for 30 min, showing a marked increase in ROSgeneration, as determined by H2DCFDA fluorescence, which was attenuatedby transplatin (1 μM).

FIG. 2B is a bar graph of the increase in NOX3 mRNA observed in UB/OC-1cells using RT-PCR, following cisplatin (5 μM) administration, which wasattenuated by prior transplatin administration at the concentrations (1,2.5, and 10 μM) shown.

FIG. 2C is a panel of photomicrographs of cochlea sections from maleWistar rats following treatment with vehicle (control) (first panel),cisplatin (11 mg/kg, i.p) (second panel), transplatin (5.5 mg/kg, i.p)followed by cisplatin (third panel), and transplatin alone (last panel),all stained for NOX3 immunoreactivity.

FIG. 3A is a bar graph of the increase in KIM-1 mRNA observed in UB/OC-1cells using RT-PCR, following cisplatin (5 μM) administration, which wasattenuated by prior transplatin administration at the concentrations (1,2.5 and 10 μM) shown.

FIG. 3B is a bar graph of the increase in TRPV1 mRNA observed in UB/OC-1cells using RT-PCR, following cisplatin (5 μM) administration, which wasattenuated by transplatin administration at the concentrations (1, 2.5and 10 μM) shown.

FIG. 3C is a panel of photomicrographs of cochlea sections from maleWistar rats following treatment with vehicle (control) (first panel),cisplatin alone (11 mg/kg, i.p) (second panel), transplatin (5.5 mg/kg,i.p) followed by cisplatin (third panel), and transplatin alone (lastpanel), all stained for KIM-1 immunoreactivity.

FIG. 4 is a panel of photomicrographs of intracellular Ca²⁺ detected byFluo-4AM and confocal microscopy, in UB/OC-1 cells treated withtransplatin (1 μM), 30 min prior to treatment with cisplatin (5 μM).

FIG. 5A is a bar graph of percentage viability of UB/OC-1 hair cellstreated with various concentrations of transplatin for 30 min prior totreatment with 20 μM cisplatin for 24 h.

FIG. 5B is a panel of photomicrographs of AT6.1 rat prostate cancercells plated at 60,000 cells per well in 24 well plates, treated with 1,2.5, 5 or 10 μM of transplatin for 30 minutes prior to treatment with100 μM cisplatin for 24 h., fixed in 4% paraformaldehyde and imaged.

FIG. 6 is a panel of photomicrographs of UB/OC-1 plated on coverslipsand treated with transplatin (1 μM) for 30 min prior to treatment withcisplatin (5 μM) for another 30 min, scanned every 6 sec by Argon laser(480 nm) exposed to FM1-43 dye on the sixth scan.

FIG. 7A is a bar graph of auditory brainstem responses (ABR) thresholdshifts (dB) obtained from naïve Wistar rats, treated with transplatin(5.5 mg/kg, i.p.), followed by cisplatin (11 mg/kg, i.p.).

FIG. 7B is a bar graph of auditory protection (no. of ears protected)observed in eight (8) naïve Wistar rats, treated with transplatin (1, 3,and 5.5 mg/kg, i.p.), followed by cisplatin (11 mg/kg, i.p.).

FIG. 7C is a panel of scanning electron photomicrographs of outer cellhair cells under control conditions (first panel), following treatmentwith cisplatin (11 mg/kg, i.p) for 72 h (second panel), followingpretreatment with transplatin (5.5 mg/kg, i.p.) followed by cisplatinadministration (third panel), and following transplatin treatment alone(last panel).

FIG. 7D is a bar graph quantifying hair cell damage seen in the basalturn of the organ of Corti following cisplatin treatment (11 mg/kg,i.p.) for 72 h, or cisplatin treatment together with transplatinpretreatment (5.5 mg/kg, i.p.).

FIG. 8A is a bar graph of the increase in mRNA expression for each ofKIM-1 (green), NOX3 (blue) and TRPV1 (red) observed in kidneys harvestedfrom male Wistar rats cells under control conditions, followingtreatment with cisplatin (11 mg/kg, i.p) for 72 h, followingpretreatment with transplatin (5.5 mg/kg) followed by cisplatinadministration, and following transplatin treatment alone.

FIG. 8B is a scatter plot of serum BUN levels in male wistar ratstreated systemically (i.p) with PBS, cisplatin (11 mg/kg), transplatin(5.5, 3 and 1 mg/kg)+Cisplatin (11 mg/kg) or transplatin (5.5 mg/kg)alone. Cisplatin treatment increases BUN values while co-administrationof transplatin with cisplatin lowers the BUN values.

FIG. 8C is a scatter plot of serum creatinine levels in male Wistar ratstreated systemically (i.p) with PBS, cisplatin (11 mg/kg), transplatin(5.5, 3 and 1 mg/kg)+Cisplatin (11 mg/kg) or transplatin (5.5 mg/kg)alone. Serum creatinine values were increased significantly withcisplatin treatment, however transplatin coadministration with cisplatinresulted in lower creatinine levels.

FIG. 9 is a bar graph of relative fluorescence intensity using H2DCFDAin dorsal root ganglion (DRG) cells from neonatal mice, over time (0 secthrough 5 min) following addition of NGF (100 ng/ml) alone (blue), andfollowing a 30 minute pretreatment with transplatin (2.5 μM) thenfollowed by NGF treatment.

FIG. 10A is a bar graph of ABR threshold shifts observed in Wistar ratsfollowing pre-treatment with vehicle or trans-tympanic transplatin (50μl of 0.5 mg/ml solution) followed by cisplatin (11 mg/kg, i.p.) over a30 min infusion period.

FIG. 10B shows scanning electron microscopy (SEM) photomicrographsshowing morphological analysis of the three rows of outer hair cells ofthe organ of Corti confirming cisplatin-induced outer hair cell damage(11 mg/kg, i.p.), which was almost completely abrogated bytrans-tympanic administration of transplatin (50 μl of 0.5 mg/mlsolution).

FIG. 10C is a bar graph quantifying outer hair cell damage/loss producedby cisplatin (11 mg/kg, i.p.), and reduction of hair cell loss/damage bypretreatment with transplatin (50 μl of 0.5 mg/ml solution).

FIG. 11 is a graph of body temperature of two rats before and aftertransplatin (Tp) treatment (5.5 mg/kg, i.p), and in a control animal(PBS), showing no apparent trend of higher body temperatures followingtransplatin administration.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

Section headings as used in this section and the entire disclosureherein are not intended to be limiting.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. For therecitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range 6-9, the numbers 7 and 8 are contemplated inaddition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitlycontemplated.

As used herein, the terms “subject” and “patient” are usedinterchangeably irrespective of whether the subject has or is currentlyundergoing any form of treatment. As used herein, the terms “subject”and “subjects” refer to any vertebrate, including, but not limited to, amammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep,hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (forexample, a monkey, such as a cynomolgous monkey, chimpanzee, etc) and ahuman). Preferably, the subject is a human.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. The meaningand scope of the terms should be clear; however, in the event of anylatent ambiguity, definitions provided herein take precedent over anydictionary or extrinsic definition. Further, unless otherwise requiredby context, singular terms shall include pluralities and plural termsshall include the singular.

In this application, the use of “or” means “and/or” unless statedotherwise. Furthermore, the use of the term “including”, as well asother forms, such as “includes” and “included”, is not limiting. Also,terms such as “element” or “component” encompass both elements andcomponents comprising one unit and elements and components that comprisemore than one subunit unless specifically stated otherwise. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those well known and commonly used in the art.

B. Transplatin Compositions

FIG. 1 is a schematic diagram of the interaction between TRPV1 and NOX3in the induction of hair cell damage or death. Data previously obtainedby the inventors (not shown) indicated that cisplatin increases TRPV1activation and expression, leading to increased intracellularaccumulation and cell death via the apoptotic or necrotic pathways. Asillustrated in FIG. 1, NOX3 generates reactive oxygen species (“ROS”),which contribute to lipid peroxidation and to damage or apoptotic celldeath. TRPV1 expression appears to be dependent on NOX3, and theactivation and expression of NOX3 appear to depend on the activity ofTRPV1. The present disclosure is based in part on the surprising findingas described herein that transplatin is protective against the toxicside effects of platinum-based chemotherapeutic agents such ascisplatin, following exposure of a cell to such an agent. Transplatinalso was unexpectedly found to have anti-inflammatory activity. Withoutbeing bound to any theory, it is believed that these effects oftransplatin may be mediated by its effect on ROS and intracellularcalcium release.

According to the present disclosure, transplatin may be used incompositions such as a pharmaceutical composition, which may be used totreat cancer in a subject or to treat inflammatory pain in a subject. Anexemplary composition includes transplatin in an amount suitable todeliver a dosage of about 0.1 to about 50 mg/kg body weight, preferablyabout 0.1 to about 20 mg/kg body weight, more preferably about 0.1 mg/kgto about 5 mg/kg body weight in a dosage form for delivery once, twice,three or four times daily, or in a dosage form for continuous deliverysuch as in a low-concentration liquid dosage form for continuous dripadministration, for example as an i.v. drip. It should be understoodthat alternative measures of dose may be used, depending on the dosageform and intended route of administration. For example, with respect tocertain dosage forms for systemic administration, dosage is oftenprovided in units of g/m2 or mg/m2. Alternatively, with respect tocertain dosage forms for localized administration, such as intra- ortrans-tympanic administration, or ear drops delivered via a ventilationtube placed in the ear, dosage is often provided as a concentration inunits of mg/ml. Exemplary, non-limiting dosages for transplatin in adosage form for systemic administration may range from about 1 mg/m2 toabout 700 mg/m2. Exemplary, non-limiting dosages for trans-tympanicadministration or as ear drops via ventilation tubes may range fromabout 0.01 mg/ml to about 100 mg/ml.

To treat cancer, transplatin can be combined in a single pharmaceuticalcomposition with a second platinum-based chemotherapy agent, oradministered sequentially either before or after administration of asecond platinum-based chemotherapy agent as detailed further herein. Aplatinum-based chemotherapy agent can be any platinum-based chemotherapyagent well known in the treatment of various cancers. While transplatinis also encompassed by the term “platinum-based chemotherapy agent”, itshould be understood that transplatin in particular is not consideredtherapeutically useful for treating cancer or any other disease, and yetexhibits the surprising protective and anti-inflammatory propertiesdisclosed herein.

It will be appreciated that the second platinum-based chemotherapy agentis preferably an agent that is demonstrated to be effective against acancer, and particularly a solid tumor. Accordingly, the secondplatinum-based chemotherapy agent is preferably though not limited tocisplatin, carboplatin, oxaliplatin, nedaplatin or nanoplatin, or anycombination thereof. In an exemplary pharmaceutical composition,transplatin is combined with cisplatin. A therapeutically effectiveamount of the second platinum-based chemotherapy agent, that is anamount that is known to be effective against a cancer that is beingtargeted in a subject, can be selected. The amount of transplatin usedcan then be calculated relative to the amount of the secondplatinum-based chemotherapy agent being used, for example by weightratio. For example, the amount of the second platinum-based chemotherapyagent can selected and the amount of transplatin used then calculatedsuch that the amount of the second platinum-based chemotherapy agent isbetween about 0.1 and about 100 times the weight of the transplatin. Thesecond platinum-based chemotherapy agent, for example cisplatin, willtypically be administered at an average of about 75-100 mg/m2 per roundof chemotherapy. A typical amount of cisplatin used in a therapeuticcomposition will therefore require between about 0.1 mg/kg and about 50mg/kg body weight of transplatin, or between about 0.1 mg/kg and about20 mg/kg body weight of transplatin, or from about 1 mg/m2 to about 700mg/m2 of transplatin, or from about 0.01 mg/ml to about 100 mg/ml oftransplatin.

The pharmaceutical compositions of the present disclosure may include a“therapeutically effective amount” of any active agent includingtransplatin and of the second platinum-based chemotherapy agent. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of transplatinmay be determined by a person skilled in the art and may vary accordingto factors such as the disease state, age, sex, and weight of theindividual, and the ability of the active agent(s) to elicit a desiredresponse in the individual. A therapeutically effective amount is alsoone in which any toxic or detrimental effects of the active agent(s) areoutweighed by the therapeutically beneficial effects. A therapeuticallyeffective amount also encompasses an amount that is effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic (i.e., preventative) result.

The pharmaceutical composition may further comprise one or moretherapeutic agents other than transplatin and a second platinum-basedchemotherapy agent. Preferably, the prophylactic or therapeutic agent(s)are known to be useful for or have been or are currently being used inthe prevention, treatment, management, or amelioration of a disorder orof one or more symptoms thereof, particularly a cancer or pain inducedby cancer. Thus the composition may further include a pharmaceuticallyacceptable carrier, diluent or excipient. As used herein,“pharmaceutically acceptable carrier, diluent or excipient” includes anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and the likethat are physiologically compatible. Examples of pharmaceuticallyacceptable carriers, diluents and excipients include but are not limitedto one or more of water, saline, phosphate buffered saline, dextrose,glycerol, ethanol and the like, as well as combinations thereof. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Pharmaceutically acceptable carriers may furthercomprise minor amounts of auxiliary substances such as wetting oremulsifying agents, preservatives or buffers, which enhance the shelflife or effectiveness of the therapeutic agent.

Supplementary active compounds can also be incorporated into thecompositions. For example, the pharmaceutical composition may furtherinclude a suitable anti-inflammatory agent such as but not limited to anon-steroidal anti-inflammatory agent, including aspirin, acetaminophen,propionic acid derivatives such as ibuprofen, naproxen, fenoprofen,ketoprofen, flurbiprofen and oxaprozin; acetic acid derivatives such asindomethacin, sulindac, etodolac, and diclofenac; enolic acidderivatives such as piroxicam, meloxicam, tenoxicam, droxicam,lornoxicam, isoxicam; fenamic acid derivatives such as mefenamic acid,meclofenamic acid, flufenamic acid, tolfenamic acid; and selective COX-2inhibitors such as celecoxib.

Various delivery systems are known and can be used to administertransplatin alone or a transplatin-containing pharmaceutical compositionas disclosed herein, including for example encapsulation in liposomes,microparticles, microcapsules, etc. Methods of administering atransplatin alone or a transplatin-containing pharmaceutical compositioninclude, but are not limited to, parenteral administration (e.g.,intradermal, intramuscular, intraperitoneal, intravenous,intraganglionic and subcutaneous), epidural administration, topicaladministration, intratumoral administration, mucosal administration(e.g., intranasal and oral routes), and aural administration (e.g.,intra- or trans-tympanic injection). Localized administration, e.g. byfocal administration onto the round window (intra-tympanic injections),or through topical ear drops such as may bedelivered via a ventilationtube placed in the ear, would limit any potential toxicity. Intra- ortrans-tympanic injections of transplatin would be beneficial inindividuals who are receiving a chemotherapeutic cocktail which includescisplatin, since this would not interfere with the therapeutic efficacyof the cisplatin. In addition, pulmonary administration can be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent. Administration can be achieved by any convenientmethod, for example by infusion or bolus injection, by absorptionthrough epithelial or mucocutaneous linings (e.g., oral mucosa, rectaland intestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

Administration can be achieved using a controlled release or sustainedrelease system. For example, a pump may be used to achieve controlled orsustained release. Polymeric materials can be used to achieve controlledor sustained release of the therapies of the present disclosure.Examples of polymers used in sustained release formulations include, butare not limited to, poly(2-hydroxy ethyl methacrylate), poly(methylmethacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate),poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides)(PLGA), and polyorthoesters. A preferred polymer for use in a sustainedrelease formulation is inert, free of leachable impurities, stable onstorage, sterile, and biodegradable. A controlled or sustained releasesystem can be placed in proximity of the prophylactic or therapeutictarget, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, in Medical Applications of Controlled Release, supra, vol. 2,pp. 115-138 (1984)).

Controlled release systems are discussed widely in the literature (for areview see Langer, Science 249:1527-1533 (1990)). Any technique known toone of skill in the art can be used to produce sustained release.

It should be understood that a pharmaceutical composition of the presentdisclosure is formulated to be compatible with its intended route ofadministration, whether the route is parenteral, e.g., intravenous,intradermal, subcutaneous, oral, intranasal (e.g., inhalation),transdermal (e.g., topical), transmucosal, trans-tympanic, rectaladministration or another accepted route of administration. Compositionsare formulated in accordance with routine procedures to prepare apharmaceutical composition adapted for intravenous, subcutaneous,intramuscular, intraganglionic, oral, intranasal, intra-aural or topicaladministration to human beings. For example, a composition forintravenous or trans-tympanic administration can be a solution insterile isotonic aqueous buffer. Where necessary, the composition mayalso include a solubilizing agent and a local anesthetic such aslidocaine to ease pain at the site of the injection.

Compositions of the present disclosure that are to be administeredtopically can be formulated in the form of an ointment, cream,transdermal patch, lotion, gel, shampoo, spray, aerosol, solution,emulsion, or other form well known to one of skill in the art. Fornon-sprayable topical dosage forms, viscous to semi-solid or solid formscomprising a carrier or one or more excipients compatible with topicalapplication and having a dynamic viscosity preferably greater than waterare typically employed. Suitable formulations include, withoutlimitation, solutions, suspensions, emulsions, creams, ointments,powders, liniments, salves, and the like, which are, if desired,sterilized or mixed with auxiliary agents (e.g., preservatives,stabilizers, wetting agents, buffers, or salts) for influencing variousproperties, such as, for example, osmotic pressure. Other suitabletopical dosage forms include sprayable aerosol preparations wherein theactive ingredient, preferably in combination with a solid or liquidinert carrier, is packaged in a mixture with a pressurized volatilecomponent (e.g., a gaseous propellant, such as freon) or in a squeezebottle. Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well known in the art.

Compositions of the present disclosure that are to be administeredtopically can be formulated in the form of an aerosol form, spray, mistor in the form of drops which may be conveniently delivered in the formof an aerosol spray presentation from pressurized packs or a nebuliser,with the use of a suitable propellant (e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas). In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges (composed of, e.g., gelatin) for use in aninhaler or insufflator may be formulated containing a powder mix of thecompound and a suitable powder base such as lactose or starch.

Compositions of the present disclosure that are to be administeredorally can be formulated in the form of tablets, capsules, cachets, gelcaps, solutions, suspensions, and the like. Tablets or capsules can beprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinised maize starch,polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose, or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc, or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell known in the art. Liquid preparations for oral or intra-auraladministration may take the form of, but not limited to, solutions,syrups or suspensions, or they may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives, or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring, and sweetening agents as appropriate. Preparations for oral orintra-aural administration may be suitably formulated for slow release,controlled release, or sustained release of a prophylactic ortherapeutic agent(s).

Pulmonary administration, e.g., by use of an inhaler or nebulizer, canbe achieved using a composition formulated with an aerosolizing agent asknown in the art.

It should also be understood that therapeutic pharmaceuticalcompositions typically must be sterile and stable under the conditionsof manufacture and storage. The composition can be formulated as asolution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile, lyophilized powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and spray drying that yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile filtered solution thereof. The proper fluidity of a solution canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including, in the composition, anagent that delays absorption, for example, monostearate salts andgelatin.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe present disclosure are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic or prophylactic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of transplatin is from about 0.1 toabout 50 mg/kg body weight, preferably about 0.1 to about 20 mg/kg bodyweight, more preferably about 0.1 to about 5 mg/kg body weight. A secondplatinum-based chemotherapy agent can be administered in an amount byweight that is between about 0.1 and about 100 times the amount byweight of transplatin. An exemplary, non-limiting range for atherapeutically or prophylactically effective amount by weight of asecond platinum-based chemotherapy agent, such as cisplatin, is betweenabout 1 and 20 times the weight of the transplatin. It is to be notedthat dosage values may vary with the type and severity of the conditionto be alleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

The present disclosure also provides a pharmaceutical composition fortreating or preventing inflammatory pain in a subject in need thereof,wherein the pharmaceutical composition comprises a therapeuticallyeffective amount of transplatin. In such composition, it will beunderstood that the therapeutically effective amount of transplatin isthe amount necessary to achieve the desired relief of pain resultingfrom an inflammatory condition, which may be a different amount formthat necessary to achieve the desired protective (e.g. otoprotective ornephroprotective) effect of transplatin observed when transplatin isadministered together with a second platinum-based chemotherapy agentthat is not transplatin (such as cisplatin). Clinical methods and testsfor determining the amount necessary to achieve the desired relief ofpain resulting from an inflammatory condition are well known.

C. Methods

The present disclosure also encompasses new methods of treatment usingtransplatin. For example, based on the unexpected protective effects oftransplatin, a method of treating or preventing ototoxic effects of anototoxic agent in a subject in need thereof includes administering tothe subject a therapeutically effective amount of transplatin. In themethod, a therapeutically effective amount of transplatin is an amountsufficient to prevent the ototoxic effects of the ototoxic agent. Thisamount may vary according to many factors as described herein aboveregarding the determination of therapeutically effective amounts. Anexemplary range for a therapeutically effective amount of transplatin inthis method is about 0.1 mg/kg body weight to about 50 mg/kg bodyweight, preferably about 0.1 mg/kg body weight to about 20 mg/kg bodyweight. A “subject in need thereof” is a subject that has been, or isexpected to be exposed to an ototoxic agent. An ototoxic agent is anyagent that causes hair cell damage, hair cell death (cell loss), hearingloss or tinnitus. Platinum-based chemotherapy agents including cisplatinare known ototoxic agents, as are aminoglycosides. Other ototoxic agentsinclude but are not limited to noise and radiation. Certain diseases andconditions are also considered ototoxic agents in the present context,such as Meniere's disease. In the method, the transplatin may beadministered to the subject before, during or after exposure of thesubject to the ototoxic agent. When administered during exposure of thesubject to an ototoxic agent that is another therapeutic agent such as aplatinum-based chemotherapy agent (e.g. cisplatin), the transplatin maybe administered simultaneously with the other therapeutic agent in asingle pharmaceutical composition as described herein above, orsimultaneously in a separate pharmaceutical composition and by adistinct route of administration. When not administered simultaneouslywith the other therapeutic agent, the transplatin may be administeredsequentially within a window of time from about 24 hours precedingadministration of the other therapeutic agent to about 24 hours afteradministration of the other therapeutic agent, including for examplesequentially within a period of about 1-6 hours either before or afteradministration of the other therapeutic agent, in a separatepharmaceutical composition. The transplatin can be administered via anyaccepted route of administration. Preferably the transplatin isadministered via intraperitoneal injection or trans-tympanic injectionin a composition suitably adapted thereto.

The present disclosure also encompasses a method of treating orpreventing nephrotoxic effects of a nephrotoxic agent in a subject inneed thereof, the method comprising administering to the subject atherapeutically effective amount of transplatin. A therapeuticallyeffective amount of transplatin is an amount sufficient to prevent thenephrotoxic effects of the nephrotxic agent. This amount may varyaccording to many factors as described herein above regarding thedetermination of therapeutically effective amounts. An exemplary rangefor a therapeutically effective amount of transplatin in this method isabout 0.1 mg/kg body weight to about 50 mg/kg body weight, preferablyabout 0.1 mg/kg body weight to about 20 mg/kg body weight. A “subject inneed thereof” is a subject that has been, or is expected to be exposedto a nephrotoxic agent. A nephrotoxic agent is any agent that causeskidney cell damage or death (cell loss). Platinum-based chemotherapyagents including cisplatin are known nephrotoxic agents, as areaminoglycosides. Other nephrotoxic agents include but are not limited todiabetes and hypertension. In the method, the transplatin may beadministered to the subject before, during or after exposure of thesubject to the nephrotoxic agent. When administered during exposure ofthe subject to a nephrotoxic agent that is another therapeutic agentsuch as a platinum-based chemotherapy agent (e.g. cisplatin), thetransplatin may be administered simultaneously with the nephrotoxicagent in a single pharmaceutical composition as described herein above,or simultaneously in a separate pharmaceutical composition and by adistinct route of administration. When not administered simultaneouslywith the other therapeutic agent, the transplatin may be administeredsequentially within a window of time from about 24 hours precedingadministration of the other therapeutic agent to about 24 hours afteradministration of the other therapeutic agent, including for examplesequentially within a period of about 1-6 hours either before or afteradministration of the other therapeutic agent, in a separatepharmaceutical composition. The transplatin can be administered forexample via any accepted route of administration. Preferably thetransplatin is administered via intraperitoneal injection.

The present disclosure also encompasses a method of treating orpreventing platinum-based chemotherapy agent-induced toxicity in asubject to be treated or treated with a platinum-based chemotherapyagent that is not transplatin, in which a therapeutically effectiveamount of transplatin is administered to the subject. In this regard, atherapeutically effective amount of transplatin is an amount sufficientto treat or prevent the platinum-based chemotherapy agent-inducedtoxicity. This amount may vary according to many factors as describedherein above regarding the determination of therapeutically effectiveamounts. An exemplary range for a therapeutically effective amount oftransplatin in this method is about 0.1 mg/kg body weight to about 50mg/kg body weight, preferably about 0.1 mg/kg body weight to about 20mg/kg body weight. A “subject in need thereof” is a subject that hasbeen, or is expected to be exposed to a platinum-based chemotherapyagent, such as for example a cancer patient. Platinum-based chemotherapyagent-induced toxicity refers to those negative side effects of suchagents including ototoxicity, nephrotoxicity, and neurotoxicity,particularly peripheral neuropathy. In the method, the transplatin maybe administered to the subject before, during or after exposure of thesubject to the platinum-based chemotherapy agent. When administeredduring exposure of the subject to a platinum-based chemotherapy agentthat is not transplatin, the transplatin may be administeredsimultaneously with the ototoxic agent in a single pharmaceuticalcomposition as described herein above, or simultaneously in a separatepharmaceutical composition and by a distinct route of administration.When not administered simultaneously with the other therapeutic agent,the transplatin may be administered sequentially within a window of timefrom about 24 hours preceding administration of the other therapeuticagent to about 24 hours after administration of the other therapeuticagent, including for example sequentially within a period of about 1-6hours either before or after administration of the other therapeuticagent, in a separate pharmaceutical composition. The transplatin can beadministered for example via any accepted route of administration.Preferably the transplatin is administered via intraperitonealinjection. Alternatively, the transplatin may be administered to thesubject after exposure of the subject to the platinum-based chemotherapyagent.

The present methods also encompass a method of treating or preventinginflammatory pain in a subject in need thereof, which includesadministering to the subject a pharmaceutical composition containing atherapeutically effective amount of transplatin. In this regard, atherapeutically effective amount of transplatin is an amount sufficientto treat or prevent inflammatory pain in the subject. This amount mayvary according to many factors as described herein above regarding thedetermination of therapeutically effective amounts. An exemplary rangefor a therapeutically effective amount of transplatin in this method isabout 0.1 mg/kg body weight to about 50 mg/kg body weight, preferablyabout 0.1 mg/kg body weight to about 20 mg/kg body weight. A “subject inneed thereof” is a subject that has been suffering, or is expected tosuffer from inflammatory pain, such as a cancer patient, or a patientsubject suffering from an inflammatory pain condition, such as but notlimited to arthritis and cancer-induced pain and chronic inflammatorypain. The transplatin may be administered simultaneously with a secondanti-inflammatory agent, such as an NSAID or combination of NSAIDS, in asingle pharmaceutical composition as described herein above, orsimultaneously in a separate pharmaceutical composition and by adistinct route of administration. The transplatin may be administeredsequentially with respect to the second anti-inflammatory agent, in aseparate pharmaceutical composition. In this method, the transplatin canbe administered via any accepted route but preferably by intraperitonealinjection, subcutaneous injection, or intraganglionic injection.

D. Adaptations of the Compositions and Methods of the Present Disclosure

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which thepresent disclosure pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The present disclosure illustratively described herein suitably may bepracticed in the absence of any element or elements, limitation orlimitations that are not specifically disclosed herein. Thus, forexample, in each instance herein any of the terms “comprising,”“consisting essentially of” and “consisting of” may be replaced witheither of the other two terms. The terms and expressions which have beenemployed are used as terms of description and not of limitation, andthere is no intention that the use of such terms and expressions excludeany equivalents of the features shown and described or portions thereof,but it is recognized that various modifications are possible within thescope of the present disclosure claimed. Thus, it should be understoodthat although the present disclosure has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

EXAMPLES

By way of example, and not of limitation, examples of the presentdisclosures shall now be given.

Example 1 Transplatin Reduces Cisplatin-Induced Reactive Oxygen Species(ROS) Generation

Imaging of ROS generation was done as previously described (P.Puntambekar et al., Essential role of Rac1/NADPH oxidase in nerve growthfactor induction of TRPV1 expression, J. N EUROCHEM. 95: 1689-1703(2005)). Briefly, UB/OC-1 cells were pretreated with transplatin and orcisplatin, and incubated with H2DCFDA dye for 30 minutes. H2DCFDAfluorescence was detected by confocal microscopy, at 30 minutesfollowing cisplatin administration. Cisplatin (2.5 μM) increases ROSgeneration in UB/OC1 cells. This increase was observed following a 30min exposure to cisplatin. FIG. 2A is a panel of photomicrographs ofUB/OC-1 cells treated with cisplatin (2.5 μM) for 30 min, showing amarked increase in ROS generation, as determined by H2DCFDAfluorescence. The observed increase in ROS generation was significantlyreduced in cells pretreated with transplatin (1 μM) (FIG. 2A). Thistransplatin concentration is in the range of cisplatin concentrationsused in vitro to produce ROS generation. Transplatin did not stimulateROS generation when administered alone. As previously observed,cisplatin induced the expression of NOX3 NADPH oxidase in the cochlea aswell as UB/OC-1 cells (D. Mukherjea et al., Expression of the kidneyinjury molecule 1 in the rat cochlea and induction by cisplatin, NEUROSCIENCE 139:733-740 (2006)).

Example 2 Transplatin Reduces Cisplatin-Induced NOX3 Generation

NOX3 expression represents a stress response by the cell in response tocisplatin. To determine whether transplatin could altercisplatin-induced NOX3 expression we used quantitative real time RT PCR.

RNA was isolated by adding 1 ml TRI reagent to 100 mg of cochlear tissueor 0.5 ml TRI reagent per well of each six well plate. Tissues werehomogenized in TRI reagent using a Polytron (setting 7, 15 sec) andcentrifuged at 12,000×g for 10 min at 4° C. The remainder of theprocedure was identical to that described in D. Mukherjea et al.,NEUROSCIENCE 139:733-740 (2006).

For real time reverse transcriptase polymerase chain reaction (RT-PCR),one microgram of total RNA was converted to cDNA using iScript cDNASynthesis Kit (Bio-Rad, Hercules, Calif.) and sample preparations wereperformed essentially as previously described (D. Mukherjea et al.,NEUROSCIENCE 139:733-740 (2006)). Amplification and detection wasperformed with the Cepheid Smart Cycler Detection System. On completionof amplification, the melting curve analysis was performed by coolingthe reaction to 60° C. and then heating slowly 95° C., according to theinstruction of manufacturer (Cepheid systems). The cycle number at whichthe sample reaches the threshold fluorescent intensity is termed thecycle threshold (Ct). The relative change in mRNA levels betweenuntreated control (1) and treated sample (2) was measured using theformula: 2-ΔΔCt (R. Soong et al., Quantitative reversetranscription-polymerase chain reaction detection of cytokeratin 20 innoncolorectal lymph nodes, C LIN. CANCER RES. 7:3423-3429 (2001)).Relative change in mRNA levels between samples was expressed as apercentage of normal control. Negative controls for both target gene andGAPDH was used for all reaction groups. Real time PCR products wereanalyzed on a 2% agarose gel to verify the correct product sizes andvisualization of the amplified product was effected using the dye SyBrGreen I (Molecular Probes, Eugene, Oreg.).

Transplatin ameliorates cisplatin induced ROS generation in UB/OC-1cells as well as Organ of Corti via NOX3 activation. FIG. 2B is a bargraph of the increase in NOX3 mRNA observed in UB/OC-1 cells usingRT-PCR, following cisplatin (5 μM) administration, with and withoutprior transplatin administration at the concentrations (1, 2.5 and 10μM) shown. Treatment of UB/OC-1 cells with transplatin 30 min prior tocisplatin administration (24 h) down regulates the increase in NOX3 mRNAas seen by real time RT-PCR. Transplatin treatment (1, 2.5 μM) aloneshowed almost no expression of NOX3 mRNA.

The effect of transplatin on cisplatin-induced NOX3 expression in maleWistar rat cochleae was investigated. Male Wistar rats were treated withvehicle (control), cisplatin (11 mg/kg, i.p), transplatin (5.5 mg/kg,i.p) followed by cisplatin, or transplatin alone. Cisplatin wasadministered in a single bolus dose. Cochleas were harvested 72 h later,fixed in 4% paraformaldehyde, decalcified, sectioned and stained forNOX3 immunoreactivity. FIG. 2C is a panel of photomicrographs of cochleasections from the rats following treatment with vehicle (control),cisplatin, transplatin followed by cisplatin, and transplatin alone, allstained for NOX3. NOX3 immunohistochemistry of mid-modiolar sections ofthe rat cochlea treated with cisplatin showed increased NOX3fluorescence in organ of Corti (OC), stria vascularis (SVA) and inspiral ganglion cells (SGC). Thus, cisplatin treatment increased NOX3protein in the outer hair cells (OHC), stria vascularis (SVA) and in thespiral ganglion (SG) cells of the organ of Corti. The cisplatin-inducedincrease in NOX3 was reduced by transplatin (5.5 mg/kg. i.p.)administration immediately before cisplatin administration. Transplatintreatment alone did not increase NOX3 expression compared to controlvehicle treated samples.

Example 3 Transplatin Decreases Cisplatin Induced KIM-1 and TRPV1Expression

Since cisplatin-stimulated ROS generation promotes the expression of twosignificant proteins, TRPV1 (P. Puntambekar et al, Essential role ofRac1/NADPH oxidase in nerve growth factor induction of TRPV1 expression,J. NEUROCHEM 95, 1689-1703 (2005)) and KIM-1 (D. Mukherjea et al.,NEUROSCIENCE 139:733-740 (2006)), quantitative real time RT PCRsubstantially as described above in Example 2 was used to determinewhether transplatin reduced the expression of these genes in cellstreated with cisplatin. UB/OC1 cells were pre-treated with transplatin(1, 2.5 and 10 μM) for 30 minutes prior to cisplatin (5 μM) treatmentfor 24 h. Total RNA was isolated, reverse transcribed and real timeRT-PCR performed. All data are presented as mean±standard error. The RTPCR data are shown in FIGS. 3A and 3B. As shown in FIG. 3A, cisplatintreatment increases KIM-1 mRNA expression by 4 fold, which increase wasattenuated to control levels by transplatin pre-treatment. As shown inFIG. 3B, TRPV1 expression increased to 3.6 fold following cisplatintreatment, which increase was abrogated by transplatin at higherconcentrations. Thus the data show that cisplatin (5 μM) treatment for24 h increased KIM-1 and TRPV1 expression in UB/OC-1 cells,respectively, by 4±0.5 fold, and by 3.5±0.7 fold, over vehicle treatedcontrols. Transplatin pre-treatment at concentrations varying from 1-10μM attenuated cisplatin-induced KIM-1 mRNA at all concentrations tested.However, higher concentrations (10 μM producing the best inhibition) oftransplatin were required for significant suppression ofcisplatin-induced TRPV1 expression, probably indicating differences insignal transduction pathways underlying the induction of these twogenes. These data are supported by KIM-1 immunocytochemistry ofmidmodiolar sections of the organ of Corti, which showed that theinduction of KIM-1 immunolabeling by cisplatin is reduced by transplatin(FIG. 3C).

Male Wistar rats were treated with vehicle (control), cisplatin (11mg/kg, i.p), transplatin (5.5 mg/kg, i.p) followed by cisplatin ortransplatin alone. Cochleas were harvested 72 h later, fixed in 4%paraformaldehyde, decalcified, sectioned and stained for KIM1immunoreactivity. FIG. 3C is a panel of photomicrographs of the cochleasections from following the different treatment conditions and stainedfor KIM-1 immunoreactivity. Cisplatin increased NOX3 immunoreactivity(FITC fluorescence) in the organ of Corti, stria vascularis and spiralganglion cells, which was decreased by transplatin. (SVA-striavascularis, OHC-outer hair cell, RM-Reissner's membrane, SG—spiralganglion).

Example 4 Transplatin Inhibits Cisplatin-Induced Intracellular Ca²⁺Release

Cisplatin induced cell death and injury is often linked to increasedintracellular Ca²⁺ release, likely via activation of TRPV1 channels,leading to induction of pro-apoptotic pathways. Previously obtained data(not shown) increased intracellular Ca²⁺ release by cisplatin in theUB/OC-1 cells (D. Mukherjea et al., Short Interfering RNA AgainstTransient Receptor Potential Vanilloid 1 Attenuates Cisplatin-InducedHearing Loss in the Rat, J. NEUROSCI. 28(49):13056-13065 (2008)).Calcium imaging was therefore used to observe transplatin effects oncisplatin-induced intracellular Ca²⁺ release. UB/OC-1 cells were grownon glass coverslips. After 30 min pre-treatment with transplatin priorto cisplatin treatment (30 minutes), cells were washed withphysiological buffer (140 mM NaCl; 4 mM KCl; 10 mM Hepes; 5 mM glucose;2 mM CaCl₂; 2 mM MgCl₂ at pH 7.4), and incubated with the calciumindicator dye, Fluo-4AM (5 μM; Invitrogen) for 30 min at 37° C. Afterincubation, cells were washed once with physiological buffer and thenimaged by a Fluoview confocal microscope (Olympus Imaging America Inc.,Center Valley, Pa., USA).

FIG. 4 is a panel of photomicrographs of intracellular Ca²⁺ detected byFluo-4AM and confocal microscopy, in UB/OC-1 cells treated withtransplatin (1 μM), 30 min prior to treatment with cisplatin (5 μM). Thenon-fluorescent images are differential interference contrast images ofthe fluorescent cells in the left fields. Cells exposed to cisplatindemonstrated increased fluorescence (detected by Fluo-4AM) by confocalmicroscopy, indicative of intracellular Ca2+ release. As shown in FIG.4, transplatin pre-treatment abrogated the cisplatin-induced increase inintracellular Ca²⁺ in the UB/OC-1 cells, likely indicating reduced TRPV1activation or cellular availability of cisplatin.

Example 5 Transplatin Prevents Cisplatin-Induced Cell Death of UB/OC-1Hair Cells

To determine whether transplatin could prevent cisplatin-induced celldeath, UB/OC-1 cells were pretreated with vehicle or transplatin (1, 2.5and 10 μM) 30 min prior to treatment with cisplatin (20 μM) for 24hours. Cells were then treated with trypan blue and the number of viablecells per high power field determined and expressed as a percent oftotal cell per field. FIG. 5A is a bar graph of the results aspercentage viability of UB/OC-1 hair cells pretreated (or not) withtransplatin before cisplatin treatment. Data are present as themean±standard error from four independent coverslips. Cisplatin reducedcell viability, as determined by trypan blue staining, to ˜20% in 24 h,i.e, ˜80% cell death. Transplatin pretreatment increased cell survivalto ˜80-97%, depending on the dose of transplatin used. Similar resultswere obtained when cell apoptosis was assessed by TUNEL staining.

Also investigated was the effect of transplatin on the ability ofcisplatin to kill tumor cells. AT6.1 rat prostate cancer cells wereplated at 60,000 cells per well in 24 well plates, and treated with 1,2.5, 5 or 10 μM of transplatin for 30 minutes prior to treatment with100 μM cisplatin for 24 h. Cells were fixed with 4% paraformaldehyde andimaged. FIG. 5B is a panel of photomicrographs of the cell images.Cisplatin treatment shows ˜80% cell loss, which does not change withtransplatin pre-treatment. Transplatin pre-treatment did not altercisplatin cytotoxicity (FIG. 5B), and transplatin alone did not causesignificant change in cell survival compared to control vehicle treatedcells.

Example 6 Transplatin Decreases the Entry of Cisplatin into UB/OC-1Cells

Recent studies have shown that cisplatin utilizes ion channels,primarily TRP channels and the purinergic receptor (P2x) ion channel forcellular entry (M. N. Rivolta et al., Auditory hair cell precursorsimmortalized from the mammalian inner ear, PROC. BIOL. SCI.265:1595-1603 (1998)). To determine whether transplatin can interactwith these channels and thereby inhibit cisplatin entry, the styryl dyeFM1-43 was used. FM1-43 is selectively taken up by cells expressing theTRP and P2x receptor channels.

UB/OC1 cells were plated on coverslips, and treated with transplatin (1μM) for 30 min prior to treatment with cisplatin (5 μM) for another 30min. Cells were scanned every 6 sec by Argon laser (480 nm) and at the6th scan FM1-43 dye was dropped on the cells. FIG. 6 is panel ofphotomicrographs of the cells following dye exposure. As shown in FIG.6, within 5 sec there is rapid uptake of the dye by the cells. Cellstreated with cisplatin alone demonstrate increased uptake of FM1-43 dye(green fluorescence), which was decreased by transplatin. Inhibition ofbasal uptake of FM1-43 was significant with transplatin but less withruthenium red. Each gray-scale image to the right of each fluorescenceimage is the differential interference constrast image for the cellsshown in the corresponding fluorescence image. Interestingly,pretreatment of cells with cisplatin (5 μM) led to an increase in entryof FM1-43 into UB/OC-1 cells, suggesting activation of the channels bycisplatin, as observed previously (D. Mukherjea et al., ShortInterfering RNA Against Transient Receptor Potential Vanilloid 1Attenuates Cisplatin-Induced Hearing Loss in the Rat, J. NEUROSCI.28(49):13056-13065 (2008)). Pre-treatment of cells with transplatin (1μM) prior to cisplatin (5 μM) administration decreased entry of the dye(as indicated by decreased fluorescence), indicating inhibition ofcisplatin entry and activation of these channels. Transplatin, addedalone, reduced fluorescence below that of the control cells, whileruthenium red (a TRPV1 antagonist) showed less inhibition of basalfluorescence than transplatin (FIG. 6). One explanation for thedifference in the effect of transplatin and ruthenium red is thattransplatin serves as an general antagonist of all TRP and P2x channels,while ruthenium red has a narrower focus by selectively targeting TRPV1.

Example 7 Transplatin Pre-Treatment Prevents Cisplatin Induced HearingLoss in Rat Model

The demonstration that transplatin could interfere with the entry ofcisplatin via TRPV1 channels in UB/OC-1 cultures prompted testing todetermine whether transplatin could ameliorate cisplatin-induced hearingloss in rat. Male Wistar rats were pre-treated with transplatin (5.5mg/kg, i.p), immediately prior to the administration of cisplatin (11mg/kg, i.p). Auditory brainstem responses (ABRs) were measured prior tocisplatin administration (pretreatment ABRs), and 72 hours followingcisplatin administration (post-treatment ABRs). FIG. 7A is a bar graphof auditory brainstem responses (ABR) threshold shifts (dB) observedunder the different treatment conditions, indicating that cisplatininduces hearing loss, as evidenced by 20-35 dB shifts in thresholds.These threshold shifts were abolished by transplatin treatment (p<0.05,N=6) at all frequencies tested. Treatment with transplatin alone did notaffect the ABR thresholds (data not shown).

Transplatin dose-response for the observed protection was determined.FIG. 7B is a bar graph of auditory protection (no. of ears protected)observed in eight (8) naïve Wistar rats, treated with transplatin (1, 3and 5.5 mg/kg, i.p.), followed by cisplatin (11 mg/kg, i.p.). Data arepresented as the mean±standard error of 8 ears examined from 4 animalsper group. As shown, dose-response for transplatin pre-treatment showedreductions in ABR thresholds in 6 of 8 rats (at the 1 mg/kg dose oftransplatin) and complete protection with 3 and 5.5 mg/kg, i.p.transplatin. Thus, transplatin produced protection in 75% of the animals(at 1 mg/kg) or complete protection (at the 3 and 5 mg/kg dose).

Scanning electron micrographs were taken of outer hair cells from ratcochleas under control conditions, following treatment with cisplatin(11 mg/kg, i.p) for 72 h, following pretreatment with transplatin (5.5mg/kg) followed by cisplatin administration, and following transplatintreatment alone. Hair cells were counted. FIG. 7C is a panel of thescanning electron photomicrographs obtained, showing that cisplatin (11mg/kg, i.p) for 72 h, induced significant hair cell damage (>40%), whiletransplatin (5.5 mg/kg) pre-treatment decreases the hair cell damagesignificantly (<5%), and transplatin alone does not cause any hair celldamage. Transplatin did not inhibit cisplatin-mediated weight loss anddid not produce weight loss by itself (data not shown).

FIG. 7D is a bar graph quantifying hair cell damage seen in the basalturn of the organ of Corti following cisplatin treatment (11 mg/kg,i.p.) for 72 h, or cisplatin treatment together with transplatinpretreatment (5.5 mg/kg, i.p.). Thus, in the basal turn of the organ ofCorti, cisplatin treatment for 72 h damaged ˜40% of outer hair cells perfield, while pretreatment with transplatin reduced this number to below10%.

Example 8 Transplatin Protects Against Cisplatin Nephrotoxicity

Nephrotoxicity is a significant toxicity associated with cisplatintherapy. To determine whether transplatin pre-treatment (5.5 mg/kg,i.p), could prevent cisplatin (11 mg/kg, i.p) induced nephrotoxicity,kidneys were harvested from male wistar rats 72 h postcisplatin/transplatin treatment. More specifically, male Wistar ratswere treated with PBS (control), cisplatin (11 mg/kg, i.p), transplatin(5.5 mg/kg, i.p) followed by cisplatin or transplatin alone for 72 h.Kidneys were harvested, total RNA was isolated, reverse transcribed andreal time RT-PCR performed substantially as described herein above. FIG.8A is a bar graph of the increase in mRNA expression for each of KIM-1(green), NOX3 (blue) and TRPV1 (red) observed. As shown in FIG. 8A,cisplatin increases KIM-1 mRNA expression by 50-fold, NOX3 expression by4 fold and TRPV1 expression by 6 fold respectively. This increase inKIM1, NOX3 and TRPV1 was attenuated to control levels by transplatin to1.1, 1.7 and 1.5 fold. Transplatin treatment (5.5 mg/kg, i.p) aloneshows KIM1, NOX3 and TRPV1 mRNA expression levels at 1.7, 1.3 and 0.5fold compared to PBS treated controls. Thus no significant changes inmRNA levels were observed following transplatin treatment alone. KIM-1molecule has been shown to be upregulated within 24 h of cisplatintreatment and is a very reliable marker for kidney injury (D. Mukherjeaet al., NEUROSCIENCE 139:733-740 (2006)). Other data (not shown)indicates that NOX3 and TRPV1 expression levels are increased incisplatin induced nephrotoxicity. Attenuation of these markers bytransplatin is indicative of its nephroprotective role.

Transplatin treatment alleviates cisplatin mediated nephrotoxicity. Malewistar rats were treated systemically (i.p) with PBS, cisplatin (11mg/kg), transplatin (5.5, 3 and 1 mg/kg)+Cisplatin (11 mg/kg) ortransplatin (5.5 mg/kg) alone. 72 hrs post treatment blood was collectedand analyzed for serum BUN (blood urea nitrogen) and serum creatininevalues. (FIG. 8B) Serum BUN values indicate that cisplatin treatmentincreases BUN values while co-administration of transplatin withcisplatin lowers the BUN values. Interestingly, transplatin treatmentalone did not raise the serum BUN values at 72 hrs. (FIG. 8C) Serumcreatinine values were increased significantly with cisplatin treatment,however transplatin coadministration with cisplatin resulted in lowercreatinine levels. Transplatin treatment alone did not change the serumcreatinine values significantly when compared to control PBS treatedvalues.

Example 9 Transplatin Demonstrates Anti-Inflammatory Activity

Recent studies have shown that ROS generation is the single mostimportant mediator of inflammation. Dorsal root ganglion (DRG) cellsisolated from neonatal mice have shown increased ROS generation inresponse to nerve growth factor (NGF; data not shown). To investigatethe possible anti-inflammatory activity of transplatin, neonatal miceDRG's were isolated, plated on coverslips and loaded with H2DCFDA (5g/ml) in PBS for 20 min, pretreated with either vehicle or transplatin(2.5 μM) and imaged by confocal microscopy with argon laser at 488 nm toobtain baseline recordings. Fluorescent images were obtained at 0 secand collected over a period of 5 min after addition of NGF (100 ng/ml).NGF generated ROS is shown by changes in fluorescence intensity overtime.

FIG. 9 is a bar graph of relative fluorescence intensity of H2DCFDA inthe neonatal mouse DRG cells, over time (0 sec through 5 min) followingaddition of NGF (100 ng/ml) alone (blue), and following a 30 minutepretreatment with transplatin. Interestingly, pre-treatment of theseDRG's with transplatin (2.5 μM) for 30 min prior to addition of NGFinhibited ROS generation (FIG. 9). This was further confirmed by usingHEK-VR1 cells (i.e., HEK-293 cells, that are stably transfected withTRPV1 receptor), wherein the ROS generation by 10 μM capsaicin (a potentVR1 agonist), was inhibited by transplatin (2.5 μM) pre-treatment (datanot shown). Thus, transplatin can be useful in the treatment ofinflammatory conditions, such as arthritis, inflammatory and chronicpain and cancer pain.

Example 10 Trans-Tympanic Transplatin Abolishes Cisplatin InducedHearing Loss in Rat Model

Pre-treatment ABRs were conducted on Wistar rats, which were thenpre-treated with vehicle or trans-tympanic transplatin (50 μl of 0.5mg/ml solution) followed by cisplatin (11 mg/kg, i.p.) over a 30 mininfusion period. Post-treatment ABRs were conducted 72 h later. FIG. 10shows the results, indicating that trans-tympanic transplatin abolishescisplatin induced hearing loss in rat model. As shown in FIG. 10A,Cisplatin produced a significant ABR threshold shift of 20-30 dB overall the 3 frequencies tested in the vehicle-pretreated groups. This wasabrogated by pretreatment with trans-tympanic transplatin. FIG. 10Bshows scanning electron photomicrographs providing a morphologicalanalysis of the three rows of outer hair cells of the organ of Corti,confirming that cisplatin produced substantial outer hair cell damage,which was almost completely abrogated by trans-tympanic administrationof transplatin. Arrows indicate areas of damage to outer hair cells.FIG. 10C is a bar graph quantifying outer hair cell damage/loss,indicating that while cisplatin produced an ˜70% hair cell damage/loss,transplatin significantly reduced the damage to ˜7% of outer hair cells.Asterisks (*) indicate statistically significant difference fromvehicle+cisplatin groups (p<0.05 by ANOVA; “TT transplatin” indicatestransplatin administered by the trans-tympanic route).

Trans-tympanic administration of drugs is the use of localizedapplication of drugs to prevent hearing loss. This route of drugadministration reduces the likehood that the drug would get into thesystemic circulation and produce side effects or cause drug-druginteractions. Since one could speculate that transplatin could interferewith the anticancer effects of cisplatin (given their structuralsimilarity), trans-tympanic administration of transplatin (by limitingits systemic availability) would eliminate this concern. The ease ofdrug delivery via the trans-tympanic route would suggest that thisprocedure could be readily performed on individuals in the out-patientsetting. These observations of otoprotection by the transtympanic routealso support the use of ventilation tubes in the tympanic membrane toallow for more a more episodic administration of transplatin in childrenprior to administering chemotherapeutic regimen containing cisplatin.

Example 11 Transplatin Treatment by the Intraperitonial Route does notIncrease Body Temperature in the Rat Model

TRPV1 antagonists were initially developed as novel treatments forinflammatory pain. However, it was observed that the administration ofclassical TRPV1 antagonists produced hyperthermia in animals and human(N. R. Gavva, Body-temperature maintenance as the predominant functionof the vanilloid receptor TRPV1. TRENDS PHARMACOL SCI 29:550-557(2007)), underscoring a role of TRPV1 in the control of bodytemperature. As such, it was of interest to examine whether transplatin,which also blocks TRPV1 (in effect a TRPV1 antagonist), would alsoproduce hyperthermia. Body temperature of the rat was noted 30 min priorto transplatin treatment (5.5 mg/kg, i.p). Body temperature was thenrecorded every 30 min for 150 min. FIG. 11 is a graph of the resultsobserved in two rats (Tp) and a control animal (PBS). No apparent trendto higher body temperatures were observed following transplatinadministration. These results from 2 rats indicate that transplatin,over the period tested, did not produce any apparent increase in bodytemperature in the rat. These results may indicate that the mechanism ofTRPV1 blockade by transplatin is different from that of classical TRPV1antagonist, and suggest that transplatin would be a safer drugalternative to classical TRPV1 antagonists.

What is claimed is:
 1. A method of treating or preventing ototoxiceffects of an ototoxic agent in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of transplatin.
 2. The method of claim 1, wherein the methodcomprises administering the transplatin to the subject before exposureof the subject to the ototoxic agent.
 3. The method of claim 1, whereinthe method comprises administering the transplatin to the subject duringexposure of the subject to the ototoxic agent.
 4. The method of claim 1,wherein the method comprises administering the transplatin to thesubject after exposure of the subject to the ototoxic agent.
 5. Themethod of claim 1, wherein the transplatin is administered viaintraperitoneal injection or trans-tympanic injection.
 6. The method ofclaim 1, wherein the transplatin is administered via oraladministration.
 7. The method of claim 1, wherein the ototoxic agent isselected from a platinum-based chemotherapy agent, radiation, Meniere'sdisease, noise, and an aminoglycoside.
 8. The method of claim 7, whereinthe ototoxic agent is noise.
 9. The method of claim 7, wherein theototoxic agent is an aminoglycoside.
 10. The method of claim 7, whereinthe ototoxic agent is a platinum-based chemotherapy agent.
 11. Themethod of claim 2, wherein the ototoxic agent is selected from aplatinum-based chemotherapy agent, radiation, Meniere's disease, noise,and an aminoglycoside.
 12. The method of claim 3, wherein the ototoxicagent is selected from a platinum-based chemotherapy agent, radiation,Meniere's disease, noise, and an aminoglycoside.
 13. The method of claim4, wherein the ototoxic agent is selected from a platinum-basedchemotherapy agent, radiation, Meniere's disease, noise, and anaminoglycoside.
 14. The method of claim 1, wherein the amount oftransplatin is between about 0.1 mg/kg and about 50 mg/kg.
 15. Themethod of claim 1, wherein the ototoxic agent is another therapeuticagent and the transplatin is administered simultaneously with the othertherapeutic agent.
 16. The method of claim 1, wherein the ototoxic agentis another therapeutic agent and the transplatin is administered withinabout 24 hours before administration of the other therapeutic agent. 17.The method of claim 1, wherein the ototoxic agent is another therapeuticagent and the transplatin is administered within about 24 hours afteradministration of the other therapeutic agent.
 18. The method of claim16, wherein the ototoxic agent is another therapeutic agent and thetransplatin is administered within about 1 to about 6 hours beforeadministration of the other therapeutic agent.
 19. The method of claim17, wherein the ototoxic agent is another therapeutic agent and thetransplatin is administered within about 1 to about 6 hours afteradministration of the other therapeutic agent.